Broad Host Range Research Articles

Genomic and Transcriptomic Diversification of Flagellin Genes Provides Insight into Environmental Adaptation and Phylogeographic Characteristics in Aeromonas hydrophila

Aeromonas hydrophila is an opportunistic motile pathogen with a broad host range, infecting both terrestrial and aquatic animals. Environmental and geographical conditions exert selective pressure on both geno- and phenotypes of pathogens. Flagellin, directly exposed to external environments and containing important immunogenic epitopes, may display significant variability in response to external conditions. In this study, we conducted a comparative analysis of ~  150 A. hydrophila genomes, leading to the identification of six subunits of the flagellin gene (fla-1 to fla-4, flaA, and flaB). Individual strains harbored different composition of flagellin subunits and copies. The composition of subunits showed distinct patterns depending on environmental sources. Strains from aquatic environments were mainly comprised of fla-1 to fla-4 subunits, while terrestrial strains predominated in groups harboring flaA and flaB subunits. Each flagellin showed varying levels of expression, with flaA and flaB demonstrating significantly higher expression compared to others. One of the chemotaxis pathways that control flagellin movement through a two-component system was significantly upregulated in flaA(+ 1)/flaB(+ 1) group, whereas flaA and flaB showed different transcriptomic expressions. The genes positively correlated with flaA expression were relevant to biofilm formation and bacterial chemotaxis, but flaB showed a negative correlation with the genes in ABC transporters and quorum sensing pathway. However, the expression patterns of fla-2 to fla-4 were identical. This suggests various types of flagellin subunits may have different biological functions. The composition and expression levels of flagellin subunits could provide valuable insights into the adaptation of A. hydrophila and the differences among strains in response to various external environments.

Identification of Phytophthora cinnamomi CRN effectors and their roles in manipulating cell death during Persea americana infection

The oomycete Phytophthora cinnamomi is a devastating plant pathogen with a notably broad host range. It is the causal agent of Phytophthora root rot (PRR), arguably the most economically important yield-limiting disease in Persea americana (avocado). Despite this, our understanding of the mechanisms P. cinnamomi employs to infect and successfully colonize avocado remains limited, particularly regarding the pathogen’s ability to maintain its biotrophic and necrotrophic lifestyles during infection. The pathogen utilises a large repertoire of effector proteins which function in facilitating and establishing disease in susceptible host plants. Crinkling and necrosis effectors (CRN/Crinklers) are suspected to manipulate cell death to aid in maintenance of the pathogens biotrophic and necrotrophic lifestyles during different stages of infection. The current study identified 25 P. cinnamomi CRN effectors from the GKB4 genome using an HMM profile and assigned putative function to them as either cell death inducers or suppressors. Function was assigned to 10 PcinCRNs by analysing their RNA-seq expression profiles, relatedness to other functionally characterised Phytophthora CRNs and tertiary protein predictions. The full-length coding sequences for these PcinCRNs were confirmed by Sanger sequencing, six of which were found to have two divergent alleles. The presence of alleles indicates that the proteins encoded may perform contradicting functions in cell death manipulation, or function in different host plant species. Overall, this study provides a foundation for future research on P. cinnamomi infection and cell death manipulation mechanisms.

Host influence on the eukaryotic virome of sympatric mosquitoes and abundance of diverse viruses with a broad host range.

Mosquitoes harbor a large diversity of eukaryotic viruses. Those viromes probably influence mosquito physiology and the transmission of human pathogens. Nevertheless, their ecology remains largely unstudied. Here, we address two key questions in virome ecology. First, we assessed the influence of mosquito species on virome taxonomic diversity and relative abundance. Contrary to most previous studies, the potential effect of the habitat was explicitly included. Thousands of individuals of Culex poicilipes and Culex tritaeniorhynchus, two vectors of viral diseases, were concomitantly sampled in three habitats over two years. A total of 95 viral taxa from 25 families were identified with meta-transcriptomics, with 75% of taxa shared by both mosquitoes. Viromes significantly differed by mosquito species but not by habitat. Differences were largely due to changes in relative abundance of shared taxa. Then, we studied the diversity of viruses with a broad host range. We searched for viral taxa shared by the two Culex species and Aedes vexans, another disease vector, present in one of the habitats. Twenty-six out of the 163 viral taxa were found in the three mosquitoes. These taxa encompassed 14 families. A database analysis supported broad host ranges for many of those viruses, as well as a widespread geographical distribution. Thus, the viromes of mosquitoes from the same genera mainly differed in the relative abundance of shared taxa, whereas differences in viral diversity dominated between mosquito genera. Whether this new model of virome diversity and structure applies to other mosquito communities remains to be determined.

Experimental Host and Vector Ranges of the Emerging Maize Yellow Mosaic Polerovirus.

Maize yellow mosaic virus (MaYMV) is an emerging polerovirus that has been detected in maize, other cereal crops, and weedy grass species in Asia, Africa, and the Americas. Disease symptoms in maize include prominent leaf tip reddening and stunting. Infection by MaYMV has been reported to reduce plant growth and yields by 10 to 30% in some instances. In this study, an experimental host range for MaYMV among agronomically important cereal crops and common grasses was established. Additional aphid species were assessed as potential vectors for MaYMV, and their transmission efficiencies were determined. Here, we report oats, foxtail millet, barley, and rye as new experimental cereal crop hosts of MaYMV in addition to confirming the previously reported hosts of corn, sorghum, wheat, and broom millet. Four of the nine other grass species evaluated were also identified as suitable experimental hosts for MaYMV: ryegrass, switchgrass, green foxtail, and sand love grass. Interestingly, no visible symptoms were present in any of the infected hosts besides the susceptible maize control. Vector range studies identified the greenbug aphid Schizaphis graminum as a new vector of MaYMV, though transmission efficiency was lower than the previously reported Rhopalosiphum maidis vector and similar to the other known aphid vector R. padi. Given MaYMV's global ubiquity, ability to evade detection, and broad host range, further characterization of yield impacts and identification of viable control strategies are desirable.

Therapeutic Advancement in Treatment and Prevention of Nipah Viral Infection: A Review

The Nipah virus (NiV) is an emerging zoonotic pathogen that poses a major risk to global health, with a high mortality rate and the potential for wide outbreaks. NiV, which originated in fruit bats, has shown a concerning potential to infect humans and cause serious respiratory and neurological disorders. The virus has a pleomorphic structure and a broad host range, preventing efforts to understand and regulate its pathogenesis. This review discusses current advances in the prevention and management of NiV, including the efficacy of several antiviral therapies in vitro and in vivo. Treatments such as Ribavirin, Favipiravir, Acyclovir, Remdesivir, and Balapiravir have produced varying results, whilst monoclonal antibodies such as m102.4, h5B3.1, and nAH1.3 provide effective neutralization but require additional testing in humans. In addition, innovative vaccination techniques such as recombinant measles virus, subunit vaccines, vector vaccines, and the novel mRNA-1215 vaccine have shown success in preclinical and early clinical trials. To reduce the risk of human transmission, preventive measures include early outbreak detection through improved surveillance systems, exposure-based screening, and educational activities. This covers vital guidelines like avoiding raw date palm sap during outbreak seasons and using physical barriers to prevent bat-to-human transmission, which is vital for controlling this deadly virus.

Partial Characterization of Three Bacteriophages Isolated from Aquaculture Hatchery Water and Their Potential in the Biocontrol of Vibrio spp.

Bacteriophages are currently considered one of the most promising alternatives to antibiotics under the ‘One Health’ approach due to their ability to effectively combat bacterial infections. This study aimed to characterize Vibrio species in hatchery water samples collected from an aquaculture farm and investigate the biocontrol potential of their bacteriophages. Vibrio spp. (n = 32) isolates confirmed by LNA probe-based qPCR were used as hosts. Three Vibrio phages were isolated. IKEM_vK exhibited a broad host range, infecting V. harveyi (n = 8), V. alginolyticus (n = 2), V. azureus (n = 1), and V. ordalii (n = 1). IKEM_v5 showed lytic activity against V. anguillarum (n = 4) and V. ordalii (n = 1), while IKEM_v14 was specific to V. scophtalmi (n = 4). The morphological appearance of phages and their lytic effects on the host were visualized using scanning electron microscopy (SEM). All three phages remained relatively stable within the pH range of 6–11 and up to 60 °C. The lytic activities and biofilm inhibition capabilities of these phages against planktonic Vibrio cells support their potential applications in controlling vibriosis in aquaculture systems.

First Report of Colletotrichum siamense Causing Anthracnose on Bixa orellana in China.

Annatto (Bixa orellana L.) is widely cultivated in China. Its seed is used as medicine and as an astringent antipyretic. Since 2019, anthracnose-type lesions have been observed on the annatto leaves in the field (about 30 hectares) in Zhanjiang (21˚18'12''N, 110˚17'22''E), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 3 ha). The early symptoms were yellow spots on the edge or tip of leaves. The spots gradually expanded and became dark brown, eventually coalescing into large irregular or circular lesions (Supplemental Figure 1-A). Ten symptomatic leaves from 10 plants were sampled. The margins of the lesions were cut into 2 × 2 mm pieces and the surfaces were disinfected with 75% ethanol for 30 sec and 2% sodium hypochlorite for 60 sec. After that, pieces were rinsed thrice in sterile water, placed on potato dextrose agar(PDA) medium, and incubated at 28 ℃ for 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (BOC-1, BOC-2, and BOC-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. Besides, the three isolates were deposited in the fungus collection at Aquatic Organisms Museum of Guangdong Ocean University. Colonies on PDA were white to gray with cottony mycelia after incubating in the dark for 6 days at 28 ℃. Conidia were one-celled, hyaline, cylindrical, clavate, and obtuse at both ends; they measured 9.6 to 18.5 µm × 3.5 to 5.5 µm (n = 50). Appressoria were oval to irregular in shape and dark brown, and they measured 6 to 9 µm × 4.5 to 8 µm (n = 30) (Supplemental Figure 1-D, E and F). These morphological characteristics matched the description of Colletotrichum siamense (Prihastuti et al. 2009;Sharma et al. 2013). For molecular identification, the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and actin (ACT) loci of the isolates were amplified using primer pairs ITS1/ITS4, GDF1/GDR1, CHS-79F/CHS-354R, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Sequences were deposited in GenBank under nos. MZ047377-MZ047379 (ITS), MZ126934-MZ1269346 (GAPDH), MZ126904-MZ1269046 (CHS-1), and MZ126844-MZ1268446 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from ITS, GAPDH, CHS-1, and ACT sequences that clustered the three isolates with C. siamense (the type strain MFLU 090230), (Supplemental Figure 2). The pathogenicity of the three isolates was tested respectively in a greenhouse maintained at 25 to 29℃ and 80% relative humidity. Annatto seeding ( n =5, 2-month-old) were inoculated with a spore solution (1 × 105 per mL) until it run-off. Whereas control plants were sprayed with sterile distilled water.. The experient was repeated three times. Anthracnose lesions were observed on the inoculated leaves after 10 days while the control plants remained healthy (Supplemental Figure 1-G, and H). The same pathogen was re-isolated from all the inoculated leaves based on morphology and ITS analysis. C. siamense has been reported to cause anthracnose in a broad range of hosts (Weir et al. 2012; Wang et al. 2017; Liu et al. 2017; Zhuo et al. 2017 ), but not in B. orellana. To our knowledge, this is the first report of C. siamense causing anthracnose on B. orellana in China. Our study provides important reference information for controlling this disease.

Botrytis cinerea detoxifies the sesquiterpenoid phytoalexin rishitin through multiple metabolizing pathways

Botrytis cinerea is a necrotrophic pathogen that infects across a broad range of plant hosts, including high-impact crop species. Its generalist necrotrophic behavior stems from its ability to detoxify structurally diverse phytoalexins. The current study aims to provide evidence of the ability of B. cinerea to tolerate the sesquiterpenoid phytoalexin rishitin, which is produced by potato and tomato. While the growth of potato pathogens Phytophthora infestans (late blight) and Alternaria solani (early blight) was severely inhibited by rishitin, B. cinerea was tolerant to rishitin. After incubation of rishitin with the mycelia of B. cinerea, it was metabolized to at least six oxidized forms. Structural analysis of these purified rishitin metabolites revealed a variety of oxidative metabolism including hydroxylation at C7 or C12, ketone formation at C5, and dihydroxylation at the 10,11-olefin. Six rishitin metabolites showed reduced toxicity to P. infestans and A. solani, indicating that B. cinerea has at least 5 distinct enzymatic reactions to detoxify rishitin. Four host-specialized phytopathogenic Botrytis species, namely B. elliptica, B. allii, B. squamosa, and B. tulipae also had at least a partial ability to metabolize rishitin as B. cinerea, but their metabolic capacity was significantly weaker than that of B. cinerea. These results suggest that the ability of B. cinerea to rapidly metabolize rishitin through multiple detoxification mechanisms could be critical for its pathogenicity in potato and tomato.

Heterologous Exchanges of Glycoprotein and Non-Virion Protein in Novirhabdoviruses: Assessment of Virulence in Yellow Perch (Perca flavescens) and Rainbow Trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses in two different species belonging to the Novirhabdovirus genus. IHNV has a narrow host range restricted to trout and salmon species, and viruses in the M genogroup of IHNV have high virulence in rainbow trout (Oncorhynchus mykiss). In contrast, the VHSV genotype IVb that invaded the Great Lakes in the United States has a broad host range, with high virulence in yellow perch (Perca flavescens), but not in rainbow trout. By using reverse-genetic systems of IHNV-M and VHSV-IVb strains, we generated six IHNV:VHSV chimeric viruses in which the glycoprotein (G), non-virion-protein (NV), or both G and NV genes of IHNV-M were replaced with the analogous genes from VHSV-IVb, and vice versa. These chimeric viruses were used to challenge groups of rainbow trout and yellow perch. The parental recombinants rIHNV-M and rVHSV-IVb were highly virulent in rainbow trout and yellow perch, respectively. Parental rIHNV-M was avirulent in yellow perch, and chimeric rIHNV carrying G, NV, or G and NV genes from VHSV-IVb remained low in virulence in yellow perch. Similarly, the parental rVHSV-IVb exhibited low virulence in rainbow trout, and chimeric rVHSV with substituted G, NV, or G and NV genes from IHNV-M remained avirulent in rainbow trout. Thus, the G and NV genes of either virus were not sufficient to confer high host-specific virulence when exchanged into a heterologous species genome. Some exchanges of G and/or NV genes caused a loss of host-specific virulence, providing insights into possible roles in viral virulence or fitness, and interactions between viral proteins.

Lysis Physiology of Pseudomonas aeruginosa Infected with ssRNA Phage PRR1.

The phage PRR1 belongs to the Leviviridae family, a group of ssRNA bacteriophages that infect Gram-negative bacteria. The variety of host cells is determined by the specificity of PRR1 to a pilus encoded by a broad host range of IncP-type plasmids that confer multiple types of antibiotic resistance to the host. Using P. aeruginosa strain PAO1 as a host, we analyzed the PRR1 infection cycle, focusing on cell lysis. PRR1 infection renders P. aeruginosa cells sensitive to lysozyme approximately 20 min before the start of a drop in suspension turbidity. At the same time, infected cells start to accumulate lipophilic anions. The on-line monitoring of the entire infection cycle showed that single-gene-mediated lysis strongly depends on the host cells' physiological state. The blockage of respiration or a reduction in the intracellular ATP concentration during the infection resulted in the inhibition of lysis. The same effect was observed when the synthesis of PRR1 lysis protein was induced in an E. coli expression system. In addition, lysis was strongly dependent on the level of aeration. Dissolved oxygen concentrations sufficient to support cell growth did not ensure efficient lysis, and a coupling between cell lysis initiation and aeration level was observed. However, the duration of the drop in suspension turbidity did not depend on the level of aeration.

Detection and genomic characterization of Klebsiella pneumoniae and Escherichia coli harboring tet(X4) in black kites (Milvus migrans) in Pakistan.

The emergence of plasmid-mediated tigecycline resistance gene tet(X4) among clinically relevant bacteria has promoted significant concerns, as tigecycline is considered a last-resort drug against serious infections caused by multidrug-resistant bacteria. We herein focused on the isolation and molecular characterization of tet(X4)-positive Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) in wild bird populations with anthropogenic interaction in Faisalabad, Pakistan. A total of 150 birds including black kites (Milvus migrans) and house crows (Corvus splendens) were screened for the presence of tigecycline resistance K. pneumoniae and E. coli. We found two K. pneumoniae and one E. coli isolate carrying tet(X4) originating from black kites. A combination of short- and long-read sequencing strategies showed that tet(X4) was located on a broad host range IncFII plasmid family in K. pneumoniae isolates whereas on an IncFII-IncFIB hybrid plasmid in E. coli. We also found an integrative and conjugative element ICEKp2 in K. pneumoniae isolate KP8336. We demonstrate the first description of tet(X4) gene in the WHO critical-priority pathogen K. pneumoniae among wild birds. The convergence of tet(X4) and virulence associated ICEKp2 in a wild bird with known anthropogenic contact should be further investigated to evaluate the potential epidemiological implications. The potential risk of global transmission of tet(X4)-positive K. pneumoniaeand E. coli warrant comprehensive evaluation and emphasizes the need for effective mitigation strategies to reduce anthropogenic-driven dissemination of AMR in the environment.

Pseudorabies virus usurps non-muscle myosin heavy chain IIA to dampen viral DNA recognition by cGAS for antagonism of host antiviral innate immunity.

Alphaherpesvirus pseudorabies virus (PRV) causes severe economic losses to the global pig industry and has garnered increasing attention due to its broad host range including humans. PRV has developed a variety of strategies to antagonize host antiviral innate immunity. However, the underlying mechanisms have not been fully elucidated. In our previous work, we demonstrated that non-muscle myosin heavy chain IIA (NMHC-IIA), a multifunctional cytoskeleton protein, attenuates innate immune responses triggered by RNA viruses. In the current study, we reported a previously unrecognized role of NMHC-IIA in counteracting PRV-induced cyclic GMP-AMP synthase (cGAS)-dependent type I interferon (IFN-I) production. Mechanistically, PRV infection led to an elevation of NMHC-IIA, strengthening the interaction between poly (ADP-ribose) polymerase 1 (PARP1) and cGAS. This interaction impeded cGAS recognition of PRV DNA and hindered downstream signaling activation. Conversely, inhibition of NMHC-IIA by Blebbistatin triggered innate immune responses and enhanced resistance to PRV proliferation both in vitro and in vivo. Taken together, our findings unveil that PRV utilizes NMHC-IIA to antagonize host antiviral immune responses via impairing DNA sensing by cGAS. This in-depth understanding of PRV immunosuppression not only provides insights for potential PRV treatment strategies but also highlights NMHC-IIA as a versatile immunosuppressive regulator usurped by both DNA and RNA viruses. Consequently, NMHC-IIA holds promise as a target for the development of broad-spectrum antiviral drugs.IMPORTANCECyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis plays a vital role in counteracting alphaherpesvirus infections. Alphaherpesviruses exploit various strategies for antagonizing cGAS-STING-mediated antiviral immune responses. However, limited examples of pseudorabies virus (PRV)-caused immunosuppression have been documented. Our findings reveal a novel role of non-muscle myosin heavy chain IIA (NMHC-IIA) in suppressing PRV-triggered innate immune responses to facilitate viral propagation both in vitro and in vivo. In detail, NMHC-IIA recruits poly (ADP-ribose) polymerase 1 (PARP1) to augment its interaction with cGAS, which impairs cGAS recognition of PRV DNA. Building on our previous demonstration of NMHC-IIA's immunosuppressive role during RNA virus infections, these findings indicate that NMHC-IIA acts as a broad-spectrum suppressor of host antiviral innate immunity in response to both DNA and RNA viruses. Therefore, NMHC-IIA will be a promising target for the development of comprehensive antiviral strategies.

Colletotrichum xishanense as a new etiological anthracnose agent on Fagopyrum gracilipes in Yunnan Province, China

Buckwheat (Fagopyrum spp.) is an important agricultural crop in some remote regions of China. In this study, two strains of Colletotrichum were isolated from brown and grey-white spots on the leaves of Fagopyrum gracilipes in Yunnan Province, China. Phylogenetic analyses inferred from combined ITS, act, chs-1, cal, gapdh, apmat, gs, and tub2 sequences revealed that these isolates are a new species, belonging to the C. gloeosporioides species complex and therefore we introduce as Colletotrichum xishanense. Pathogenicity tests indicated that C. xishanense infects not only herbaceous plant but also woody plant. Given the broader host range and stronger pathogenicity, it is time to focus on disease management and resistance breeding.

Genetic and Functional Diversity Help Explain Pathogenic, Weakly Pathogenic, and Commensal Lifestyles in the Genus Xanthomonas.

The genus Xanthomonas has been primarily studied for pathogenic interactions with plants. However, besides host and tissue-specific pathogenic strains, this genus also comprises nonpathogenic strains isolated from a broad range of hosts, sometimes in association with pathogenic strains, and other environments, including rainwater. Based on their incapacity or limited capacity to cause symptoms on the host of isolation, nonpathogenic xanthomonads can be further characterized as commensal and weakly pathogenic. This study aimed to understand the diversity and evolution of nonpathogenic xanthomonads compared to their pathogenic counterparts based on their cooccurrence and phylogenetic relationship and to identify genomic traits that form the basis of a life history framework that groups xanthomonads by ecological strategies. We sequenced genomes of 83 strains spanning the genus phylogeny and identified eight novel species, indicating unexplored diversity. While some nonpathogenic species have experienced a recent loss of a type III secretion system, specifically the hrp2 cluster, we observed an apparent lack of association of the hrp2 cluster with lifestyles of diverse species. We performed association analysis on a large data set of 337 Xanthomonas strains to explain how xanthomonads may have established association with the plants across the continuum of lifestyles from commensals to weak pathogens to pathogens. Presence of distinct transcriptional regulators, distinct nutrient utilization and assimilation genes, transcriptional regulators, and chemotaxis genes may explain lifestyle-specific adaptations of xanthomonads.

Machine-learning-guided Directed Evolution for AAV Capsid Engineering.

Target gene delivery is crucial to gene therapy. Adeno-associated virus (AAV) has emerged as a primary gene therapy vector due to its broad host range, long-term expression, and low pathogenicity. However, AAV vectors have some limitations, such as immunogenicity and insufficient targeting. Designing or modifying capsids is a potential method of improving the efficacy of gene delivery, but hindered by weak biological basis of AAV, complexity of the capsids, and limitations of current screening methods. Artificial intelligence (AI), especially machine learning (ML), has great potential to accelerate and improve the optimization of capsid properties as well as decrease their development time and manufacturing costs. This review introduces the traditional methods of designing AAV capsids and the general steps of building a sequence-function ML model, highlights the applications of ML in the development workflow, and summarizes its advantages and challenges.

Isolation of a PRD1-like phage uncovers the carriage of three putative conjugative plasmids in clinical Burkholderia contaminans

The Burkholderia cepacia complex (Bcc) is a group of increasingly multi-drug resistant opportunistic bacteria. This resistance is driven through a combination of intrinsic factors and the carriage of a broad range of conjugative plasmids harbouring virulence determinants. Therefore, novel treatments are required to treat and prevent further spread of these virulence determinants. In the search for phages infective for clinical Bcc isolates, CSP1 phage, a PRD1-like phage was isolated. CSP1 phage was found to require pilus machinery commonly encoded on conjugative plasmids to facilitate infection of Gram-negative bacteria genera including Escherichia and Pseudomonas. Whole genome sequencing and characterisation of one of the clinical Burkholderia isolates revealed it to be Burkholderia contaminans. B. contaminans 5080 was found to contain a genome of over 8 Mbp encoding multiple intrinsic resistance factors, such as efflux pump systems, but more interestingly, carried three novel plasmids encoding multiple putative virulence factors for increased host fitness, including antimicrobial resistance. Even though PRD1-like phages are broad host range, their use in novel antimicrobial treatments shouldn’t be dismissed, as the dissemination potential of conjugative plasmids is extensive. Continued survey of clinical bacterial strains is also key to understanding the spread of antimicrobial resistance determinants and plasmid evolution.

Genetic toolbox for Photorhabdus and Xenorhabdus: pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling

BackgroundBacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work.ResultsIn the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743.ConclusionsThe results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.

Detection of vesivirus in minks (Neovison vison), Italy 2021.

Vesiviruses are important animal pathogens with a broad host range, and they have also been involved in accidental contamination of cells used for the production of drugs for rare and life-threatening human diseases. A vesivirus (family Caliciviridae) was detected in minks (Neovison vison) with respiratory and neurological signs, during syndromic surveillance for SARS-CoV-2 conducted in Italy. The complete genome (8,397 nucleotides in length) of the vesivirus strain ITA/2021/mink/TE (OR130287) was obtained by combining NGS approach with 5' and 3' RACE protocols. The virus was seemingly more related (95.9-97.2% nt identity in the partial RNA-dependent RNA polymerase) to American vesivirus isolates 9/1980/US, 12/1980/US, and 20/1980/US dating back to the early 1980s than to recent mink strains. These results highlight the importance of gathering information on the virome of animals.

Procymidone Application Contributes to Multidrug Resistance of Botrytis cinerea.

The necrotrophic pathogen Botrytis cinerea infects a broad range of plant hosts and causes substantial economic losses to many crops. Although resistance to procymidone has been observed in the field, it remains uncertain why procymidone is usually involved in multidrug resistance (MDR) together with other fungicides. Nine mutants derived from the B. cinerea strain B05.10 through procymidone domestication exhibited high resistance factors (RFs) against both procymidone and fludioxonil. However, the fitness of the mutants was reduced compared to their parental strain, showing non-sporulation and moderate virulence. Furthermore, the RFs of these mutants to other fungicides, such as azoxystrobin, fluazinam, difenoconazole, and pyrimethanil, ranged from 10 to 151, indicating the occurrence of MDR. Transcriptive expression analysis using the quantitative polymerase chain reaction (qPCR) revealed that the mutants overexpressed ABC transporter genes, ranging from 2 to 93.7-fold. These mutants carried single-point mutations W647X, R96X, and Q751X within BcBos1 by DNA sequencing. These alterations in BcBos1 conferred resistance to procymidone and other fungicides in the mutants. Molecular docking analysis suggested distinct interactions between procymidone and Bos1 in the B. cinerea standard strain B05.10 or the resistant mutants, suggesting a higher affinity of the former towards binding with the fungicide. This study provides a comprehensive understanding of the biological characteristics of the resistant mutants and conducts an initial investigation into its fungicide resistance traits, providing a reference for understanding the causes of multidrug resistance of B. cinerea in the field.

Genomic and Phenotypic Analysis of Salmonella enterica Bacteriophages Identifies Two Novel Phage Species.

Bacteriophages (phages) are potential alternatives to chemical antimicrobials against pathogens of public health significance. Understanding the diversity and host specificity of phages is important for developing effective phage biocontrol approaches. Here, we assessed the host range, morphology, and genetic diversity of eight Salmonella enterica phages isolated from a wastewater treatment plant. The host range analysis revealed that six out of eight phages lysed more than 81% of the 43 Salmonella enterica isolates tested. The genomic sequences of all phages were determined. Whole-genome sequencing (WGS) data revealed that phage genome sizes ranged from 41 to 114 kb, with GC contents between 39.9 and 50.0%. Two of the phages SB13 and SB28 represent new species, Epseptimavirus SB13 and genera Macdonaldcampvirus, respectively, as designated by the International Committee for the Taxonomy of Viruses (ICTV) using genome-based taxonomic classification. One phage (SB18) belonged to the Myoviridae morphotype while the remaining phages belonged to the Siphoviridae morphotype. The gene content analyses showed that none of the phages possessed virulence, toxin, antibiotic resistance, type I-VI toxin-antitoxin modules, or lysogeny genes. Three (SB3, SB15, and SB18) out of the eight phages possessed tailspike proteins. Whole-genome-based phylogeny of the eight phages with their 113 homologs revealed three clusters A, B, and C and seven subclusters (A1, A2, A3, B1, B2, C1, and C2). While cluster C1 phages were predominantly isolated from animal sources, cluster B contained phages from both wastewater and animal sources. The broad host range of these phages highlights their potential use for controlling the presence of S. enterica in foods.